This invention relates to the preparation of lecithin:cholesterol acyltransferase. In particular, this invention is concerned with the expression of lecithin:cholesterol acyltransferase in recombinant host cell culture.
Cholesterol of peripheral origin is transported through the plasma for catabolism in the liver. The sequence of reactions involved in this pathway (reverse cholesterol transport) is believed to be important in peripheral cholesterol homeostatis (1). A key component in the process of transport and metabolism of cholesterol in plasma is its esterification by lecithin:cholesterol acyltransferase. This enzyme, a glycoprotein of apparent molecular weight .about.63,000 (2,3), is secreted from the liver into the plasma compartment (4,5). Under physiological conditions, lecithin:cholesterol acyltransferase catalyzes the transfer of acyl groups from the sn2 position of lecithin to the 3-OH of free cholesterol. Apolipoprotein A-I (apoA-I), the major protein of plasma high density lipoprotein (HDL) is a potent activator of lecithin:cholesterol acyltransferase activity (6). The conversion of diffusible cholesterol to its insoluble ester form in plasma is important in maintaining a concentration gradient between cell membranes and plasma. When lecithin:cholesterol acyltransferase activity is inhibited in vitro or in vivo, or is genetically defective, cholesterol is no longer transported to plasma, and accumulates in the tissues (7-9). While the lecithin:cholesterol acyltransferase protein has been purified to homogeneity by several laboratories, little is known of its structure of mechanism of action. Furthermore, purification from natural sources is expensive and there is a risk of contamination in the final product from adventitious viruses and the like. In addition, the activity of natural lecithin:cholesterol acyltransferase, i.e., lecithin:cholesterol acyltransferase having an amino acid sequence identical to that of lecithin:cholesterol acyltransferase present in a given animal species (or its naturally-occurring alleles), is not readily modified. Accordingly, it is an object herein to provide a method for the economical preparation of lecithin:cholesterol acyltransferase free of infective contamination and which is sufficiently flexible to enable the preparation of lecithin:cholesterol acyltransferase amino acid sequence variants having properties that differ from those of natural lecithin:cholesterol acyltransferase.